Expression of Escherichia coli homoserine kinase in mouse 3T3 cells.
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ABSTRACT:
SUBMITTER: Rees WD
PROVIDER: S-EPMC1130769 | biostudies-other | 1992 Feb
REPOSITORIES: biostudies-other
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Project description:We have subcloned the coding sequence for the Escherichia coli threonine synthase gene into a eukaryotic expression vector based on the simian-virus-40 early promoter. When mouse 3T3 cells which already expressed homoserine kinase were transfected with the new plasmid, the cells were able to incorporate radioactivity from [14C]homoserine into their cell proteins. Stable cell lines were established by co-transfecting 3T3 cells with the plasmid coding for threonine synthase and another coding for homoserine kinase and G-418 (Geneticin) resistance. Cells were selected for G-418 resistance and then screened for an ability to synthesize threonine from homoserine and incorporate it into the cell protein. A cell line which expressed both the homoserine kinase and threonine synthase genes was capable of growth in a threonine-deficient medium containing homoserine.
| S-EPMC1132519 | biostudies-other
Project description:Quorum sensing networks have been identified in over one hundred bacterial species to date. A subset of these networks regulate group behaviors, such as bioluminescence, virulence, and biofilm formation, by sending and receiving small molecules called homoserine lactones (HSLs). Bioengineers have incorporated quorum sensing pathways into genetic circuits to connect logical operations. However, the development of higher-order genetic circuitry is inhibited by crosstalk, in which one quorum sensing network responds to HSLs produced by a different network. Here, we report the construction and characterization of a library of ten synthases including some that are expected to produce HSLs that are incompatible with the Lux pathway, and therefore show no crosstalk. We demonstrated their function in a common lab chassis, Escherichia coli BL21, and in two contexts, liquid and solid agar cultures, using decoupled Sender and Receiver pathways. We observed weak or strong stimulation of a Lux receiver by longer-chain or shorter-chain HSL-generating Senders, respectively. We also considered the under-investigated risk of unintentional release of incompletely deactivated HSLs in biological waste. We found that HSL-enriched media treated with bleach were still bioactive, while autoclaving deactivates LuxR induction. This work represents the most extensive comparison of quorum signaling synthases to date and greatly expands the bacterial signaling toolkit while recommending practices for disposal based on empirical, quantitative evidence.
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Project description:Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator that exerts numerous biological activities both as a receptor ligand and as an intracellular second messenger. In the present study, we explored roles of sphingosine kinase (SphK), an S1P-producing enzyme, in adipose tissue. We utilized mouse 3T3-L1 cells as an in vitro model of adipogenesis, using a mixture of insulin/dexamethasone/3-isobutyl-1-methylxanthine (IBMX) to induce differentiation. Real-time quantitative PCR (qRT-PCR) assays revealed that the expression levels of transcripts encoding both isoforms of SphK-1 and SphK-2 are up-regulated during adipogenesis (37.6- and 6.6-fold vs. basal, P < 0.05, respectively). Concomitantly, SphK-1/SphK-2 protein abundance and S1P contents of these cells increased at 3 days after hormonal stimulation. Loss-of-function approaches by pharmacological inhibition of SphK activity as well as by transfection with small interfering RNA (siRNA) against SphK-1 led to significant attenuation of lipid droplet accumulation and adipocyte marker gene expression. We detected marked elevation of SphK-1 mRNA in adipose tissue derived from 13-week-old ob/ob mice with obese phenotype than their lean littermates. These results suggest that increased expression of SphK, an S1P-producing enzyme, plays a significant role during adipogenesis, potentially providing a novel point of control in adipose tissue.
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Project description:BackgroundQuorum sensing (QS) networks are more commonly known as acyl homoserine lactone (HSL) networks. Recently, p-coumaroyl-HSL has been found in a photosynthetic bacterium. p-coumaroyl-HSL is derived from a lignin monomer, p-coumaric acid, rather than a fatty acyl group. The p-coumaroyl-HSL may serve an ecological role in diverse QS pathways between p-coumaroyl-HSL producing bacteria and specific plants. Interference with QS has been regarded as a novel way to control bacterial infections. Heterologous production of the QS molecule, p-coumaroyl-HSL, could provide a sustainable and controlled means for its large-scale production, in contrast to the restricted feedback regulation and extremely low productivity of natural producers.ResultsWe developed an artificial biosynthetic process for phenylacetyl-homoserine lactone analogs, including cinnamoyl-HSL, p-coumaroyl-HSL, caffeoyl-HSL, and feruloyl-HSL, using a bioconversion method via E. coli (CB1) in the co-expression of the codon-optimized LuxI-type synthase (RpaI) and p-coumaroyl-CoA ligase (4CL2nt). In addition to this, we show the de novo production of p-coumaroyl-HSL in heterologous host E. coli (DN1) and tyrosine overproducing E. coli (DN2), containing the rpaI gene in addition to p-coumaroyl-CoA biosynthetic genes. The yields for p-coumaroyl-HSL reached 93.4 ± 0.6 and 142.5 ± 1.0 mg/L in the S-adenosyl-L-methionine and L-methionine feeding culture in the DN2 strain, respectively.ConclusionsThis is the first report of a de novo biosynthesis in a heterologous host yielding a QS molecule, p-coumaroyl-HSL from a glucose medium using a single vector system combining p-coumaroyl-CoA biosynthetic genes and the LuxI-type synthase gene.
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Project description:l-Homoserine, which is one of the few amino acids that is not produced on a large scale by microbial fermentation, plays a significant role in the synthesis of a series of valuable chemicals. In this study, systematic metabolic engineering was applied to target Escherichia coli W3110 for the production of l-homoserine. Initially, a basic l-homoserine producer was engineered through the strategies of overexpressing thrA (encoding homoserine dehydrogenase), removing the degradative and competitive pathways by knocking out metA (encoding homoserine O-succinyltransferase) and thrB (encoding homoserine kinase), reinforcing the transport system, and redirecting the carbon flux by deleting iclR (encoding the isocitrate lyase regulator). The resulting strain constructed by these strategies yielded 3.21 g/liter of l-homoserine in batch cultures. Moreover, based on CRISPR-Cas9/dCas9 (nuclease-dead Cas9)-mediated gene repression for 50 genes, the iterative genetic modifications of biosynthesis pathways improved the l-homoserine yield in a stepwise manner. The rational integration of glucose uptake and recovery of l-glutamate increased l-homoserine production to 7.25 g/liter in shake flask cultivation. Furthermore, the intracellular metabolic analysis further provided targets for strain modification by introducing the anaplerotic route afforded by pyruvate carboxylase to oxaloacetate formation, which resulted in accumulating 8.54 g/liter l-homoserine (0.33 g/g glucose, 62.4% of the maximum theoretical yield) in shake flask cultivation. Finally, a rationally designed strain gave 37.57 g/liter l-homoserine under fed-batch fermentation, with a yield of 0.31 g/g glucose.IMPORTANCE In this study, the bottlenecks that sequentially limit l-homoserine biosynthesis were identified and resolved, based on rational and efficient metabolic-engineering strategies, coupled with CRISPR interference (CRISPRi)-based systematic analysis. The metabolomics data largely expanded our understanding of metabolic effects and revealed relevant targets for further modification to achieve better performance. The systematic analysis strategy, as well as metabolomics analysis, can be used to rationally design cell factories for the production of highly valuable chemicals.
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Project description:Glycine N-acyltransferase (GLYAT) is a phase II metabolic detoxification enzyme for exogenous (xenobiotic) and endogenous carboxylic acids; consisting of fatty acids, benzoic acid, and salicylic acid. GLYAT catalyzes the formation of hippurate (N-benzoylglycine) from the corresponding glycine and benzoyl-CoA. Herein, we report the successful expression, purification, and characterization of recombinant mouse GLYAT (mGLYAT). A 34kDa mGLYAT protein was expressed in Escherichia coli and purified to homogeneity by nickel affinity chromatography to a final yield of 2.5mg/L culture. Characterization for both amino donors and amino acceptors were completed, with glycine serving as the best amino donor substrate, (kcat/Km)app=(5.2±0.20)×10(2)M(-1)s(-1), and benzoyl-CoA serving as the best the amino acceptor substrate, (kcat/Km)app=(4.5±0.27)×10(5)M(-1)s(-1). Our data demonstrate that mGLYAT will catalyzed the chain length specific (C2-C6) formation of N-acylglycines. The steady-state kinetic constants determined for recombinant mGLYAT for the substrates benzoyl-CoA and glycine, were shown to be consistent with other reported species (rat, human, bovine, ovine, and rhesus monkey). The successful recombinant expression and purification of mGLYAT can lead to solve unanswered questions associated with this enzyme, consisting of what is the chemical mechanism and what catalytic residues are essential for the how this phase II metabolic detoxification enzyme conjugates glycine to xenobiotic and endogenous carboxylic acids.
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Project description:Recently, more studies have aimed at identifying selective peroxisome proliferator-activated receptor gamma (PPARγ) modulators that transactivate the expression of PPARγ-dependent genes as partial agonists to improve diabetic symptoms with fewer side effects compared to classic PPARγ agonists such as thiazolidinediones. We found that dihydrosanguinarine (DHS) treatment induced preadipocyte differentiation and lipid droplet accumulation in 3T3-L1 cells, but this effect is weaker than that elicited by the full PPARγ agonist troglitazone. Furthermore, this effect was reduced by the addition of a PPARγ antagonist, indicating the involvement of PPARγ signaling. Our results suggest that the stimulatory effects of DHS on adipocyte differentiation and insulin sensitivity are mediated by suppressing adenosine monophosphate-activated protein kinase (AMPK) alpha, upregulating the expression of PPARγ and its target genes (particularly Glut-4 and adiponectin) and reducing PPARγ phosphorylation. DHS significantly enhanced the glucose uptake in 3T3-L1 adipocytes without observable cytotoxicity at the effective concentration (5 μM) applied.
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Project description:In Escherichia coli, growth is limited at elevated temperatures mainly because of the instability of a single enzyme, homoserine o-succinyltransferase (MetA), the first enzyme in the methionine biosynthesis pathway. The metA gene from the thermophile Geobacillus kaustophilus cloned into the E. coli chromosome was found to enhance the growth of the host strain at elevated temperature (44 degrees C), thus confirming the limited growth of E. coli due to MetA instability. In order to improve E. coli growth at higher temperatures, we used random mutagenesis to obtain a thermostable MetA(E. coli) protein. Sequencing of the thermotolerant mutant showed five amino acid substitutions: S61T, E213V, I229T, N267D, and N271K. An E. coli strain with the mutated metA gene chromosomally inserted showed accelerated growth over a temperature range of 34 to 44 degrees C. We used the site-directed metA mutants to identify two amino acid residues responsible for the sensitivity of MetA(E. coli) to both heat and acids. Replacement of isoleucine 229 with threonine and asparagine 267 with aspartic acid stabilized the protein. The thermostable MetA(E. coli) enzymes showed less aggregation in vivo at higher temperature, as well as upon acetic acid treatment. The data presented here are the first to show improved E. coli growth at higher temperatures solely due to MetA stabilization and provide new knowledge for designing E. coli strains that grow at higher temperatures, thus reducing the cooling cost of bioprocesses.
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Project description:Conjugation is a key mechanism for horizontal gene transfer and plays an important role in bacterial evolution, especially with respect to antibiotic resistance. However, little is known about the role of donor and recipient cells in regulation of conjugation. Here, using an Escherichia coli (SM10??)-Pseudomonas aeruginosa (PAO1) conjugation model, we demonstrated that deficiency of lasI/rhlI, genes associated with generation of the quorum sensing signals N-acyl homoserine lactones (AHLs) in PAO1, or deletion of the AHLs receptor SdiA in the donor SM10?? both facilitated conjugation. When using another AHLs-non-producing E. coli strain EC600 as recipient cells, deficiency of sdiA in donor SM10?? hardly affect the conjugation. More importantly, in the presence of exogenous AHLs, the conjugation efficiency between SM10?? and EC600 was dramatically decreased, while deficiency of sdiA in SM10?? attenuated AHLs-inhibited conjugation. These data suggest the conjugation suppression function of AHLs-SdiA chemical signaling. Further bioinformatics analysis, ?-galactosidase reporter system and electrophoretic mobility shift assays characterized the binding site of SdiA on the promoter region of traI gene. Furthermore, deletion of lasI/rhlI or sdiA promoted traI mRNA expression in SM10?? and PAO1 co-culture system, which was abrogated by AHLs. Collectively, our results provide new insight into an important contribution of quorum sensing system AHLs-SdiA to the networks that regulate conjugation.
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Project description:In Escherichia coli, RecA-single-stranded DNA (RecA-ssDNA) filaments catalyze DNA repair, recombination, and induction of the SOS response. It has been shown that, while many (15 to 25%) log-phase cells have RecA filaments, few (about 1%) are induced for SOS. It is hypothesized that RecA's ability to induce SOS expression in log-phase cells is repressed because of the potentially detrimental effects of SOS mutagenesis. To test this, mutations were sought to produce a population where the number of cells with SOS expression more closely equaled the number of RecA filaments. Here, it is shown that deleting radA (important for resolution of recombination structures) and increasing recA transcription 2- to 3-fold with a recAo1403 operator mutation act independently to minimally satisfy this condition. This allows 24% of mutant cells to have elevated levels of SOS expression, a percentage similar to that of cells with RecA-green fluorescent protein (RecA-GFP) foci. In an xthA (exonuclease III gene) mutant where there are 3-fold more RecA loading events, recX (a destabilizer of RecA filaments) must be additionally deleted to achieve a population of cells where the percentage having elevated SOS expression (91%) nearly equals the percentage with at least one RecA-GFP focus (83%). It is proposed that, in the xthA mutant, there are three independent mechanisms that repress SOS expression in log-phase cells. These are the rapid processing of RecA filaments by RadA, maintaining the concentration of RecA below a critical level, and the destabilizing of RecA filaments by RecX. Only the first two mechanisms operate independently in a wild-type cell.
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